FIELD OF THE INVENTION
[0001] Disclosed are methods and compositions for producing isoprenoids such as squalene
using yeast.
BACKGROUND OF THE INVENTION
[0002] The following description of the background of the invention is provided simply as
an aid in understanding the invention and is not admitted to describe or constitute
prior art to the invention.
[0003] Isoprenoids, such as squalene, are commercially important types of lipids. They have
excellent lubricity, oxidative stability, low pour points, low freezing points, high
flash points, and facile biodegradability. Squalene is currently produced by extraction
from olive oil or cold water shark liver oil at a high unit cost. Because of the high
unit cost, economically feasible uses for squalene and squalane (the fully hydrogenated
derivative of squalene) are in small market applications such as watch lubricants,
pharmaceuticals/nutraceuticals, cosmetics, perfumes and as chemical intermediates
for high-value products.
[0004] There exist, however, significant potential markets for biodegradable lubricants,
lubricant additives, and hydraulic fluids. Biodegradability of these products is particularly
important for environmentally sensitive applications, such as agricultural applications,
or where considerable lubricant or hydraulic fluids may be lost to the environment.
The potential markets for biodegradable lubricants, lubricant additives, and hydraulic
fluids are quite large, estimated to be on the order of five million metric tons per
annum.
[0005] Biodegradable lubricants, lubricant additives, and hydraulic fluids derived from
vegetable and animal fats and oils are available, but they have drawbacks. They typically
solidify at relatively high temperatures (i.e., they solidify in cold weather) and
have flash points that are too low for use in hot conditions, (i.e., they break down
or combust under normal hot engine conditions).
[0006] Thus, a cost effective method of production of squalene is desired that would allow
for largc-scalc manufacturing and widespread use of squalene and squalane in biodegradable
lubricants, lubricant additives, and hydraulic fluids.
[0007] Chang et al., (Appl. Microbiol., Biotechnol., 2008, 78, 963-72) discloses the discovery of a wild type yeast,
Pseudozyma sp. JCC207, that produces "
a large amount of squalene and several polyunsaturated fatty acids." Chang et al. describe isolating
Pseudozyma sp. JCC207 from seawater collected near Guam, USA, and are unsure whether
Pseudozyma sp. JCC207 is a new species or a variant of
P. regulosa or
P. aphidis. In the article, "the efficiency of squalene production [of
Pseudozyma sp. JCC207] was investigated under different conditions."
[0008] Dow AgroSciences LLC,
Using Yeast Fermentation to Produce Cost-Fffective and Biodegradable Lubricants, http://statusreports.atp.nist.gov/reports/95-01-0148PDF.pdf, discloses that "[t]he
company proposed to use genetic engineering to alter the metabolic characteristics
of an oleaginous (oily) yeast to increase the yeast's ability to produce isoprenes
through biosynthesis." Specifically, four enzymes were targeted: ACCase, hydroxymcthylglutaryl
CoA reductase (HMGR), squalene synthetase, and squalene epoxidase.
[0009] U.S. Patent No. 5,460,949 discloses "[a] method increasing the accumulation of squalene and specific sterols
in yeast." In particular, it is disclosed that "[s]qualene and sterol accumulation
is increased by increasing the expression level of a gene encoding a polypeptide having
the HMG-CoA reductase activity."
[0010] WO 2008/130372 A9 relates to methods for the biological production of certain sterol compounds, and
to systems for producing oleaginous yeasts or fungi that are capable of producing
certain sterol compounds.
SUMMARY OF THE INVENTION
[0011] Subject matter of the present invention is a composition as defined in claim 1, and
a method of producing squalene as defined in claim 2. The dependent claims relate
to particular embodiments thereof.
[0012] One aspect of the present invention accordingly relates to a composition comprising
a genetically converted yeast. The genetically converted yeast expresses one or more
modified enzymes having one or more designed mutations, wherein the one or more designed
mutations are at defined positions within said enzyme. The one or more modified enzymes
comprise HMG-CoA reductase, and the HMG-CoA reductase has increased activity and/or
expression. The yeast produces increased quantities of squalene as compared to the
native yeast, and it is a
Yarrowia lipolytica strain selected from the group consisting of ATCC 90812, ATCC MYA-2613, or Yeastern
polg.
[0013] Another aspect of the present invention relates to a method of producing squalene
by a genetically converted yeast. The method comprises increasing or decreasing activity
or expression of one or more enzymes in the isoprenoid biosynthesis pathway. The enzyme
activity or expression is increased or decreased by one or more designed mutations,
wherein the one or more designed mutations are at defined positions within said enzyme.
The one or more modified enzymes comprise HMG-CoA reductase, and the HMG-CoA reductase
has increased activity and/or expression. The yeast produces increased quantities
of squalene as compared to the native yeast, and it is a
Yarrowia lipolytica strain selected from the group consisting of ATCC 90812, ATCC MYA-2613, or Yeastern
polg.
[0014] According to a particular embodiment, the genetically converted yeast is derived
from an oleaginous yeast.
[0015] According to a particular embodiment, the one or more modified enzymes further comprise
a modified enzyme selected from the group consisting of acetyl-CoA carboxylase ("ACCase"),
squalene epoxidase, squalene synthase, ATP citrate lyase, ATP citrate synthase, mevalonate
kinase, glycerol kinase and 5-aminolevulinate synthase.
[0016] According to a particular embodiment, the activity or expression is increased at
least 1.2-fold; or 1.5-fold; or 2-fold; or 3- fold; or 4- fold; or 5- fold; or 10-fold;
or 10-fold; or 20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold; or
100,000-fold; or 1,000,000-fold higher than the activity or expression of the corresponding
native yeast.
[0017] According to a particular embodiment, the yeast is the Yeastern polg strain of
Yarrowia lipolytica.
[0018] According to a particular embodiment, an antifungal agent is present in the composition
or is added to the yeast in the method. For example, an antifungal agent is present
in the composition or is added to the yeast in the method at a concentration between
0.5 to 100 µg/ml, between 1 to 25 µg/ml, or between 10 to 15 µg/ml. According to a
particular embodiment, the antifungal agent may be an allylamine antifungal agent.
For example, the antifungal agent may be an allylamine antifungal agent present at
a concentration between 0.5 to 100 µg/ml, such as between 1 to 25 µg/ml, or between
10 to 15 µg/ml. According to a particular embodiment, the antifungal agent may be
terbinafine. For example, the terbinafine may be present at a concentration between
0.5 to 100 µg/ml, such as between 1 to 25 µg/ml, or between 10 to 15 µg/ml.
[0019] According to a particular embodiment, the method comprises cultivating the yeast
with an antifungal agent; wherein the yeast is the Yeastern polg strain of
Yarrowia lipolytica and wherein the antifungal agent is terbinafine. For example, the terbinafine may
be present at a concentration of 12.5 µg/ml or greater.
[0020] As disclosed herein, increased amounts of an isoprenoid (for example, squalene) produced
by a genetically converted or non-genetically converted yeast may be the result of
mutating, modifying and/or altering the activity of one or more enzymes within the
isoprenoid biosynthesis pathway. For example acetyl-CoA carboxylase (or "ACCase"),
HMG-CoA reductase, squalene epoxidase, squalene synthase, ATP citrate synthase, mevalonate
kinase (e.g.,
Y. lipolytica mevalonate kinase (Genolevures YAL10B16038g)), glycerol kinase (e.g.,
Y. lipolytica glycerol kinase (Genolevures YALI0F00484g)) and/or 5-aminolevulinate synthase (e.g.,
encoded by
Saccharomyces cerevisiae HEM1 gene) may be modified, mutated or have altered activity.
[0021] As disclosed herein, a genetically converted yeast expressing a modified enzyme may
be produced by introducing a mutation in the enzyme through use of a gene repair oligonucleobase
as described herein. Such methods may include introducing a gene repair oligonucleobase
containing a specific mutation for a target gene of interest into a yeast cell by
any of a number of methods well-known in the art (e.g., electroporation, LiOAc, biolistics,
spheroplasting, and/or
Agrobacterium (see, for example,
McClelland, C.M., Chang, Y.C., and Kwon-Chung, K.J. (2005) Fungal Genetics and Biology
42:904-913) and identifying a cell having the mutated enzyme.
[0022] As disclosed herein, a method of producing isoprenoids, preferably squalene, may
include providing a genetically converted or non-genetically converted yeast as described
herein and extracting squalene from the yeast. The method may include exposing yeast
(either genetically converted or non-genetically converted) to an antifungal agent
(for example, an allylamine antifungal agent such as terbinafine) and extracting squalene
from the yeast. The method may include exposing a genetically converted yeast such
as described herein to an antifungal agent (for example, an allylamine antifungal
agent such as terbinafine) and extracting squalene from the yeast. The method may
include exposing a non-genetically converted yeast such as described herein to an
antifungal agent (for example, an allylamine antifungal agent such as terbinafine)
and extracting squalene from the yeast.
[0023] In the methods and compositions disclosed herein that include an antifungal agent
(for example, an allylamine antifungal agent such as terbinafine), the antifungal
agent (for example terbinafine or other antifungal agent) may be added or present
in a concentration at or above about 1 µg/ml; or about 5 (µg/ml; or about 10 µg/ml;
or about 11 µg/ml; or about 12 µg/ml; or about 12.5 µg/ml; or about 13 µg/ml; or about
15 µg/ml; or about 16 µg/ml; or about 20 µg/ml; or about 25 µg/ml; or about 30 µg/ml;
or about 40 µg/ml; or about 50 µg/ml or greater. In the methods and compositions disclosed
herein that include an antifungal agent (for example, an allylamine antifungal agent
such as terbinafine), the antifungal agent (for example terbinafine or other antifungal
agent) may be added or present in a concentration between about 0.5 to 100 µg/ml;
or 0.5 to 50 µg/ml; or 1 to 50 µg/ml; or 5 to 50 µg/ml; or 8 to 50 µg/ml; or 10 to
50 µg/ml; or 12 to 50 µg/ml; or 15 to 50 µg/ml; or 15 to 50 µg/ml; or 25 to 50 µg/ml;
or 1 to 25 µg/ml; or 5 to 25 µg/ml; or 10 to 25 µg/ml; or 10 to 20 µg/ml; or 10 to
15 µg/ml.
[0024] Disclosed herein is a genetically converted yeast that produces isoprenoids. In certain
examples, the genetically converted yeast produces squalene.
[0025] Further disclosed herein is a genetically converted yeast, wherein the yeast is genetically
converted such that it produces increased levels of squalene as compared to the corresponding
native yeast. In certain examples, the genetically converted yeast expresses one or
more modified enzymes having one or more mutations. In certain examples the expression
level of one or more enzymes in the genetically converted yeast is increased or decreased
relative to the corresponding native yeast. In related examples, the genetically converted
yeast expresses one or more modified enzymes having one or more mutations and the
expression level of one or more enzymes in the genetically converted yeast is increased
or decreased relative to the corresponding native yeast. In certain preferred examples
a genetically converted yeast as disclosed herein is genetically converted by introducing
a mutation into an enzyme using a gene repair oligobase. In some examples a genetically
converted yeast as disclosed herein is genetically converted by introducing one or
more mutations at or around the translation start site of a gene encoding an enzyme
to increase or decrease expression of the enzyme, for example, as described in
US Patent Application Nos. 10/411,969 and
11/625,586. In certain examples, the enzyme modified in a genetically converted yeast as disclosed
herein includes one or more enzymes selected from the group consisting of acetyl-CoA
carboxylase (or "ACCase"), HMG-CoA reductase, squalene epoxidase, squalene synthase,
ATP citrate lyase, ATP citrate synthase, mevalonate kinase (e.g.,
Y. lipolytica mevalonate kinase (Genolevures YALI0B16038g)), glycerol kinase (e.g.,
Y. lipolytica glycerol kinase (Genolevures YALI0F00484g)) and 5-aminolcvulinate synthasc.
[0026] A nucleobase comprises a base, which is a purine, pyrimidine, or a derivative or
analog thereof. Nucleosides are nucleobases that contain a pentosefuranosyl moiety,
e.g., an optionally substituted riboside or 2'-deoxyriboside. Nucleosides can be linked
by one of several linkage moieties, which may or may not contain phosphorus. Nucleosides
that are linked by unsubstituted phosphodiester linkages are termed nucleotides. "Nucleobases"
as used herein include peptide nucleobases, the subunits of peptide nucleic acids,
and morpholine nucleobases as well as nucleosides and nucleotides.
[0027] An oligonucleobase is a polymer of nucleobases, which polymer can hybridize by Watson-Crick
base pairing to a DNA having the complementary sequence. An oligonucleobase chain
has a single 5' and 3' terminus, which are the ultimate nucleobases of the polymer.
A particular oligonucleobase chain can contain nucleobases of all types. An oligonucleobase
compound is a compound comprising one or more oligonucleobase chains that are complementary
and hybridized by Watson-Crick base pairing. Nucleobases are either deoxyribo-type
or ribo-type. Ribo-type nucleobases are pentosefuranosyl containing nucleobases wherein
the 2' carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type
nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases
that do not contain a pentosefuranosyl moiety.
[0028] An oligonucleobase strand generically includes both oligonucleobase chains and segments
or regions of oligonucleobase chains. An oligonucleobase strand has a 3' end and a
5' end. When an oligonucleobase strand is coextensive with a chain, the 3' and 5'
ends of the strand are also 3' and 5' termini of the chain.
[0029] The term "gene repair oligonucleobase" is used herein to denote oligonucleobases,
including mixed duplex oligonucleotides, non-nucleotide containing molecules, single
stranded oligodeoxynucleotides and other gene repair molecules as described in detail
below.
[0030] In some
examples, a genetically converted yeast or non-genetically converted
yeast as disclosed herein is derived from an oleaginous yeast. In certain preferred
examples, a genetically converted yeast or non-genetically converted yeast as disclosed herein
is derived from a yeast selected from the group consisting of
Cryptococcus curvatus, Yarrowia lipolytica, Rhodotorula glutinus, and
Rhorosporidium toruloides. In some preferred
examples, the genetically converted yeast or non-genetically converted yeast is derived from
a yeast selected from the group consisting of
Cryptococcus curvatus, Yarrowia lipolytica, and
Rhodotorula glutinus. In related
examples, the genetically converted yeast or non-genetically converted yeast is derived from
a yeast selected from the group consisting of
Cryptococcus curvatus, and
Rhodotorula glutinus. In certain preferred
examples, the genetically converted yeast or non-genetically converted yeast is not derived
from
Yarrowia lipolytica. The genetically converted yeast used in the methods and compositions according to the present invention is a
Yarrowia lipolytica strain selected from the group consisting of ATCC 90812, ATCC MYA-2613, and Yeastern
polg.
[0031] In certain preferred
examples, an enzyme that is modified in a genetically
converted yeast as disclosed herein is acetyl-CoA carboxylase (or "ACCase"). In some preferred
examples, acetyl-CoA carboxylase in a genetically converted yeast is modified such that its
activity and/or expression is decreased relative to the corresponding native yeast;
or such that the activity and/or expression is eliminated. In other
examples, the acetyl-CoA carboxylase may be modified so that its substrate selectivity is altered.
In some preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of acetyl-CoA carboxylase is reduced relative to the corresponding native yeast but
the activity is not eliminated. In some preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of acetyl-CoA carboxylase in the genetically converted yeast is reduced to about 90%;
or about 80%; or about 70%; or about 60%; or about 50%; or about 40%; or about 30%;
or about 20%; or about 10%; or about 5% of the activity and/or expression of the corresponding
native yeast. In related
examples, the genetically converted yeast is modified such that the activity and/or expression
of acetyl-CoA carboxylase in the genetically converted yeast is between about 90-95%;
or about 80-90%; or about 70-80%; or about 60-70%; or about 50-60%; or about 40-50%;
or about 30-40%; or about 20-30%; or about 10-20%; or about 5-10%; or about 2-5% of
the activity and/or expression of the corresponding native yeast.
[0032] An enzyme that is modified in the genetically converted yeast used in the methods
and compositions according to the present invention is HMG-CoA reductase. In some
preferred examples, HMG-CoA reductase in a genetically converted yeast is modified such that its activity
and/or expression is increased relative to the corresponding native yeast. In other
examples, the HMG-CoA reductase may be modified so that it substrate selectivity is altered.
In certain preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of HMG-CoA reductase in the genetically converted yeast is increased to at least 1.2-fold;
or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 15-fold; or
20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold;
or 1,000,000-fold higher than the activity and/or expression of the corresponding
native yeast.
[0033] In certain preferred
examples, an enzyme that is modified in a genetically converted yeast as
disclosed herein is squalene epoxidase. In some preferred examples squalene epoxidase in a genetically converted yeast is modified such that its activity
and/or expression is decreased relative to the corresponding native yeast; or such
that the activity and/or expression is eliminated. In other
examples, the squalene epoxidase may be modified so that its substrate selectivity is altered.
In some preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of squalene epoxidase is reduced relative to the corresponding native yeast but the
activity is not eliminated. In certain
examples, the squalene epoxidase is modified to include one or more mutations or homologs of
one or more mutations associated with increased sensitivity to terbinafine. In certain
examples, the yeast is not
Saccharomyces cerevisiae and the squalene epoxidase is modified to include the homologs of one or more of
the following mutations associated with increased sensitivity to terbinafine in the
Saccharomyces cerevisiae ERG1 gene: G30S, L37P, and R269G (see, e.g., Tumowsky, 2005, 2007 and 2008). In certain
examples, the yeast is Y. lipolytica and the squalene epoxidase is modified to include the
homologs of one or more of the following mutations associated with increased sensitivity
to terbinafine in the
Saccharomyces cerevisiae ERG1 gene: G30S, L37P, and R269G (see, e.g., Turnowsky, 2005, 2007 and 2008). In
some
examples, the yeast squalene epoxidase gene is modified as described herein by synthesis and
replacement of the wild-type gene or by introduction of mutations by RTDS. In some
preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of squalene epoxidase in the genetically converted yeast is reduced to about 90%;
or about 80%; or about 70%; or about 60%; or about 50%; or about 40%; or about 30%;
or about 20%; or about 10%; or about 5% of the activity and/or expression of the corresponding
native yeast. In related
examples, the genetically converted yeast is modified such that the activity and/or expression
of squalene epoxidase in the genetically converted yeast is between about 90-95%;
or about 80-90%; or about 70-80%; or about 60-70%; or about 50-60%; or about 40-50%;
or about 30-40%; or about 20-30%; or about 10-20%; or about 5-10%; or about 2-5% of
the activity and/or expression of the corresponding native yeast.
[0034] In certain preferred
examples, an enzyme that is modified in a genetically converted yeast as
disclosed herein is squalene synthase. In some preferred examples squalene synthase in a genetically converted yeast is modified such that its activity
and/or expression is increased relative to the corresponding native yeast. In other
examples, the squalene synthase may be modified so that it substrate selectivity is altered.
In certain preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of squalene synthase in the genetically converted yeast is increased to at least 1.2-fold;
or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold; or 10-fold; or 15-fold; or
20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold;
or 1,000,000-fold higher than the activity and/or expression of the corresponding
native yeast.
[0035] In certain preferred
examples, an enzyme that is modified in a genetically
converted yeast as disclosed herein is ATP citrate lyase. In some
examples, either or both subunits of ATP citrate lyase genes (for example,
Yarrowia lipolytica ATP citrate lyase; Genoleveres YALI0D24431g and YALI0E34793g) are modified as described
herein. In certain
examples, the activity of ATP citrate lyase in a modified yeast is increased by the insertion
and/or heterologous expression of an animal ATP lyase gene which comprises a single
subunit holoenzyme. In some preferred
examples, ATP citrate lyase in a genetically converted yeast is modified such that its activity
and/or expression is increased relative to the corresponding native yeast. In certain
preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of ATP citrate lyase in the genetically converted yeast is increased to at least 1.2-fold;
or 1.5-fold; or 2-fold; or 3-fold; or 4- fold; or 5- fold; or 10-fold; or 10-fold;
or 20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold; or 100,000-fold;
or 1,000,000-fold higher than the activity and/or expression of the corresponding
native yeast.
[0036] In certain
examples of the compositions and methods disclosed herein, the enzyme that is modified in
a genetically converted yeast or non-genetically converted yeast is ATP citrate synthase.
Preferably, its activity and/or expression is increased relative to the corresponding
native yeast.
[0037] In certain preferred
examples, an enzyme that is modified in a genetically
converted yeast as disclosed herein is mevalonate kinase (e.g.,
Y .lipolytica mevalonate kinase (Genolevures YALI0B16038g)). In some preferred
examples, nevalonate kinase (e.g.,
Y.
lipolytica mevalonate kinase (Genolevures YALI0B16038g)) in a genetically converted yeast is
modified such that its activity and/or expression is increased relative to the corresponding
native yeast. In certain preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of mevalonate kinase (e.g.,
Y.
lipolytica mevalonate kinase (Genolevures YALI0B16038g)) in the genetically converted yeast
is increased to at least 1.2-fold; or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or
5-fold; or 10-fold; or 15-fold; or 20-fold; or 50-fold; or 100-fold; or 1,000-fold;
or 10,000-fold; or 100,000-fold; or 1,000,000-fold higher than the activity and/or
expression of the corresponding native yeast.
[0038] In certain preferred
examples, an enzyme that is modified in a genetically
converted yeast as disclosed herein is glycerol kinase (e.g.,
Y. lipolytica glycerol kinase (Genolevures YALI0F00484g)). In some preferred
examples, glycerol kinase (e.g.,
Y .lipolytica glycerol kinase (Genolevures YALI0F00484g)) in a genetically converted yeast is modified
such that its activity and/or expression is increased relative to the corresponding
native yeast. In certain preferred
examples, the genetically converted yeast is modified such that the activity and/or expression
of mevalonate kinase glycerol kinase (e.g.,
Y.
lipolytica glycerol kinase (Genolevures YAL10F00484g)) in the genetically converted yeast is
increased to at least 1.2-fold; or 1.5-fold; or 2-fold; or 3-fold; or 4-fold; or 5-fold;
or 10-fold; or 15-fold; or 20-fold; or 50-fold; or 100-fold; or 1,000-fold; or 10,000-fold;
or 100,000-fold; or 1,000,000-fold higher than the activity and/or expression of the
corresponding native yeast.
[0039] In certain
examples of the compositions and methods
disclosed herein, the enzyme that is modified in a genetically converted yeast or non-genetically converted
yeast is 5-aminolevulinate synthase (e.g., encoded by
Saccharomyces cerevisiae HEM1 gene). Preferably, its activity and/or expression is increased relative to the
corresponding native yeast.
[0040] In certain preferred
examples disclosed herein, the converted yeast is a genetically converted yeast; in other preferred
examples, the genetically converted yeast is a transgenic yeast. Further
examples are a yeast that includes both transgenic and genetic alterations.
[0041] In certain
examples, disclosed are compositions that include a yeast (for example a genetically converted yeast
such as disclosed herein or a non-genetically converted yeast) wherein at least 10%
of the total lipid content is squalene; or at least 20% of the total lipid content
is squalene; or at least 25% of the total lipid content is squalene; or at least 28%
of the total lipid content is squalene; or at least 30% of the total lipid content
is squalene; or at least 32% of the total lipid content is squalene; or at least 35%
of the total lipid content is squalene; or at least 37% of the total lipid content
is squalene; or at least 38% of the total lipid content is squalene; or at least 40%
of the total lipid content is squalene; or at least 42% of the total lipid content
is squalene; or at least 45% of the total lipid content is squalene; or at least 47%
of the total lipid content is squalene; or at least 50% of the total lipid content
is squalene; or at least 52% of the total lipid content is squalene; or at least 55%
of the total lipid content is squalene; or at least 57% of the total lipid content
is squalene; or at least 60% or more of the total lipid content is squalene.
[0042] The phrase "genetically converted yeast" or "genetically altered yeast" as used herein
refers to a yeast having one or more genetic modifications, such as transgenes and/or
modified enzymes which contain one or more designed mutation(s). Such designed mutations
may result in a modified enzyme having an activity that is different from the native
enzyme. Such differences can include differences in substrate specificity or level
of activity. As used herein, a "transgenic yeast" is one type of a "genetically converted
yeast".
[0043] The term "native yeast" as used herein refers to a yeast that is not genetically
converted (i.e., transgenic or genetically altered). Native yeasts include wild type
yeasts as well as yeasts that have been selectively bred to attain particular characteristics.
[0044] The phrase "transgenic yeast" refers to a yeast having a gene from another yeast
species or non-yeast species. Such a gene may be referred to as a "transgene."
[0045] As used herein the term "target gene" refers to the gene encoding the enzyme to be
modified.
[0046] The phrase "oleaginous yeast" refers to a yeast that contains at least about 20%
cell dry weight (cdw) lipid extractable from the organism. The capacity to accumulate
levels of lipid at least about 20% cdw is not confined to a particular genus; greater
than about 20% cdw lipid has been reported in
Lipomyces lipofer, L. starkeyi, L. tetrasporus, Candida lipolytica, C. diddensiae,
C. paralipolytica, C. curvata, Cryptococcus albidus, Cryptococcus laurentii, Geotrichum
candidum, Rhodotorula graminus, Trichosporon pullulans,
Rhodosporidium toruloides,
Rhodotorula glutinus,
Rhodotorula gracilis, and
Yarrowia lipolytica. See, e.g.,
Tatsumi, et al. U.S. Pat. No. 4,032,405, and
Rattray, Microbial Lipids, Vol. 1 (1998).
[0047] The term "about" as used herein means in quantitative terms plus or minus 10%. For
example, "about 3%" would encompass 2.7-3.3% and "about 10%" would encompass 9-11%.
[0048] Unless otherwise indicated, any percentages stated herein are percent by weight.
[0049] Other features and advantages of the invention will be apparent from the following
description of the preferred embodiments and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
Types of Yeast.
[0050] The compositions and methods as disclosed herein can be based on any of a number
of yeast species or strains. In certain
examples, the yeast is an oleaginous yeast. For example the yeast may be
Cryptococcus curvatus (for example ATCC 20508),
Yarrowia lipolytica (for example ATCC 20688 or ATCC 90811),
Rhodotorula glutinus (for example ATCC 10788 or ATCC 204091), and
Rhorosporidium toruloides. The inventors have discovered that, relative to certain other yeast (such as
Yarrowia lipolytica)
, Cryptococcus curvatus and
Rhodotorula glutinis grow to very high cell densities on a wide variety of substrates, and produce large
amounts of total lipid under many culture conditions. Accordingly, in certain
examples Cryptococcus curvatus and
Rhodotorula glutinis may be particularly advantageous for the compositions and methods as disclosed herein.
There are many genetic tools (for example, transformation protocols, selectable markers)
that are well developed and specific for
Yarrowia lipolytica; as such in some embodiments
Yarrowia lipolytica may be particularly advantageous for the compositions and methods as disclosed
herein. In the compositions and methods according to the present invention, the genetically
converted yeast is a Yarrowia lipolytica strain selected from the group consisting
of ATCC 90812, ATCC MYA-2613, and Yeastern po1g.
Gene repair oligonucleobases
[0051] The methods disclosed herein may be practiced with "gene repair oligonucleobases"
having the conformations and chemistries as described in detail below. The "gene repair
oligonucleobases" include mixed duplex oligonucleotides, non-nucleotide containing
molecules, single stranded oligodeoxynucleotides and other gene repair molecules described
in the below noted patents and patent publications. The "gene repair oligonucleobases"
have also been described in published scientific and patent literature using other
names including "recombinagenic oligonucleobases;" "RNA/DNA chimcric oligonucleotides;"
"chimeric oligonucleotides;" "mixed duplex oligonucleotides (MDONs);" "RNA DNA oligonucleotides
(RDOs);" "gene targeting oligonucleotides;" "genoplasts;" "single stranded modified
oligonucleotides;" "Single stranded oligodeoxynucleotide mutational vectors;" "duplex
mutational vectors;" and "heteroduplex mutational vectors."
[0053] The gene repair oligonucleobases in Kmiec I and/or Kmiec II contain two complementary
strands, one of which contains at least one segment of RNA-type nucleotides (an "RNA
segment") that are base paired to DNA-type nucleotides of the other strand.
[0054] Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can
be substituted for nucleotides. Additional gene repair molecules that can be used
are described in
U.S. Pat. Nos. 5,756,325;
5,871,984;
5,760,012;
5,888,983;
5,795,972;
5,780,296;
5,945,339;
6,004,804; and
6,010,907 and in International Patent No.
PCT/US00/23457; and in International Patent Publication Nos.
WO 98/49350;
WO 99/07865;
WO 99/58723;
WO 99/58702; and
WO 99/40789 .
[0055] In one
example, the gene repair oligonucleobase is a mixed duplex oligonucleotide in which the RNA-type
nucleotides of the mixed duplex oligonucleotide are made RNase resistant by replacing
the 2'-hydroxyl with a fluoro, chloro or bromo functionality or by placing a substituent
on the 2'-O. Suitable substituents include the substituents taught by the Kmiec II.
Alternative substituents include the substituents taught by
U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications
EP 629 387 and
EP 679 657 (collectively, the Martin Applications).
[0056] As used herein, a 2'-fluoro, chloro or bromo derivative of a ribonucleotide or a
ribonucleotide having a 2'-OH substituted with a substituent described in the Martin
Applications or Sproat is termed a "2'-Substituted Ribonucleotide." As used herein
the term "RNA-type nucleotide" means a 2'-hydroxyl or 2'-Substitutcd Nucleotide that
is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted
phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec
II. As used herein the term "deoxyribo-type nucleotide" means a nucleotide having
a 2'-H, which can be linked to other nucleotides of a gene repair oligonucleobase
by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught
by Kmiec I or Kmiec II.
[0057] In a particular
example disclosed herein, the gene repair oligonucleobase is a mixed duplex oligonucleotide that is linked
solely by unsubstituted phosphodiester bonds. In alternative
examples, the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based
linkages as taught by Kmiec II. In yet another
example, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2'-Substituted
Nucleotide. Particular preferred
examples of 2'-Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy,
2'-hydroxylethyloxy, 2'-methoxyethyloxy, 2'-fluoropropyloxy and 2'-trifluoropropyloxy
substituted ribonucleotides. More preferred
examples of 2'-Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy,
and 2'-allyloxy substituted nucleotides. In another
example, the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.
[0058] Although mixed duplex oligonucleotides having only a single type of 2'-substituted
RNA-type nucleotide are more conveniently synthesized, the methods
disclosed herein can be practiced with mixed duplex oligonucleotides having two or more types of RNA-type
nuclcotides. The function of an RNA segment may not be affected by an interruption
caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides,
accordingly, the term RNA segment encompasses such as "interrupted RNA segment." An
uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative
example, an RNA segment can contain alternating RNase-resistant and unsubstituted 2'-OH nucleotides.
The mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more
preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and
second strands are Watson-Crick base paired. In one
example, the strands of the mixed duplex oligonucleotide are covalently bonded by a linker,
such as a single stranded hexa, penta or tetranucleotide so that the first and second
strands are segments of a single oligonucleotide chain having a single 3' and a single
5' end. The 3' and 5' ends can be protected by the addition of a "hairpin cap" whereby
the 3' and 5' terminal nucleotides are Watson-Crick paired to adjacent nucleotides.
A second hairpin cap can, additionally, be placed at the junction between the first
and second strands distant from the 3' and 5' ends, so that the Watson-Crick pairing
between the first and second strands is stabilized.
[0059] The first and second strands contain two regions that are homologous with two fragments
of the target gene, i.e., have the same sequence as the target gene. A homologous
region contains the nucleotides of an RNA segment and may contain one or more DNA-type
nucleotides of connecting DNA segment and may also contain DNA-type nucleotides that
are not within the intervening DNA segment. The two regions of homology are separated
by, and each is adjacent to, a region having a sequence that differs from the sequence
of the target gene, termed a "heterologous region." The heterologous region can contain
one, two or three mismatched nucleotides. The mismatched nucleotides can be contiguous
or alternatively can be separated by one or two nucleotides that are homologous with
the target gene. Alternatively, the heterologous region can also contain an insertion
or one, two, three or of five or fewer nucleotides. Alternatively, the sequence of
the mixed duplex oligonucleotide may differ from the sequence of the target gene only
by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex
oligonucleotide. The length and position of the heterologous region is, in this case,
deemed to be the length of the deletion, even though no nucleotides of the mixed duplex
oligonucleotide are within the heterologous region. The distance between the fragments
of the target gene that are complementary to the two homologous regions is identically
the length of the heterologous region when a substitution or substitutions is intended.
When the heterologous region contains an insertion, the homologous regions are thereby
separated in the mixed duplex oligonucleotide farther than their complementary homologous
fragments are in the gene, and the converse is applicable when the heterologous region
encodes a deletion.
[0060] The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous
region, i.e., a region that is identical in sequence to a fragment of the target gene,
which segments together preferably contain at least 13 RNA-type nucleotides and preferably
from 16 to 25 RNA-type nucleotides or yet more preferably 18-22 RNA-type nucleotides
or most preferably 20 nucleotides. In one
example, RNA segments of the homology regions are separated by and adjacent to, i.e., "connected
by" an intervening DNA segment. In one example, each nucleotide of the heterologous
region is a nucleotide of the intervening DNA segment. An intervening DNA segment
that contains the heterologous region of a mixed duplex oligonucleotide is termed
a "mutator segment."
[0061] In another
example disclosed herein, the gene repair oligonucleobase is a single stranded oligodeoxynucleotide mutational
vector (SSOMV), which is disclosed in International Patent Application
PCT/US00/23457,
U.S. Pat. Nos. 6,271,360,
6,479,292, and
7,060,500 . The sequence of the SSOMV is based on the same principles as the mutational vectors
described in
U.S. Pat. Nos. 5,756,325;
5,871,984;
5,760,012;
5,888,983;
5,795,972;
5,780,296;
5,945,339;
6,004,804; and
6,010,907 and in International Publication Nos.
WO 98/49350;
WO 99/07865;
WO 99/58723;
WO 99/58702; and
WO 99/40789. The sequence of the SSOMV contains two regions that are homologous with the target
sequence separated by a region that contains the desired genetic alteration termed
the mutator region. The mutator region can have a sequence that is the same length
as the sequence that separates the homologous regions in the target sequence, but
having a different sequence. Such a mutator region can cause a substitution. Alternatively,
the homologous regions in the SSOMV can be contiguous to each other, while the regions
in the target gene having the same sequence are separated by one, two or more nucleotides.
Such a SSOMV causes a deletion from the target gene of the nucleotides that are absent
from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous
regions may be adjacent in the target gene but separated by one two or more nucleotides
in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of
target gene.
[0062] The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified
phosphodiester bonds except that the 3' terminal and/or 5' terminal internucleotide
linkage or alternatively the two 3' terminal and/or 5' terminal internucleotide linkages
can be a phosphorothioate or phosphoamidate. As used herein an internucleotide linkage
is the linkage between nucleotides of the SSOMV and does not include the linkage between
the 3' end nucleotide or 5' end nucleotide and a blocking substituent, see supra.
In a specific
example the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of
the homology regions are, accordingly, a total length of at least 20 deoxynucleotides
and at least two homology regions should each have lengths of at least 8 deoxynucleotides.
[0063] The SSOMV can be designed to be complementary to either the coding or the non-coding
strand of the target gene. When the desired mutation is a substitution of a single
base, it is preferred that both the mutator nucleotide be a pyrimidine. To the extent
that is consistent with achieving the desired functional result it is preferred that
both the mutator nucleotide and the targeted nucleotide in the complementary strand
be pyrimidines. Particularly preferred are SSOMV that encode transversion mutations,
i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide
in the complementary strand.
[0064] In addition to the oligodeoxynucleotide the SSOMV can contain a 5' blocking substituent
that is attached to the 5' terminal carbons through a linker. The chemistry of the
linker is not critical other than its length, which should preferably be at least
6 atoms long and that the linker should be flexible. A variety of non-toxic substituents
such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent
dye can be used. Particularly preferred as reagents to make SSOMV are the reagents
sold as Cy3™ and Cy5™ by Glen Research, Sterling Va., which are blocked phosphoroamidites
that upon incorporation into an oligonucleotide yield 3,3,3',3'-tetramethyl N,N'-isopropyl
substituted indomonocarbocyanine and indodicarbocyaninc dyes, respectively. Cy3 is
the most preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be
conveniently linked to the 5' terminal of the oligodeoxynucleotide through as a phosphodiester
with a 5' terminal phosphate. The chemistry of the dye linker between the dye and
the oligodeoxynucleotide is not critical and is chosen for synthetic convenience.
When the commercially available Cy3 phosphoramidite is used as directed the resulting
5' modification consists of a blocking substituent and linker together which are a
N-hydroxypropyl, N'-phosphatidylpropyl 3,3,3',3'-tetramethyl indomonocarbocyanine.
[0065] In one preferred
example, the indocarbocyanine dye is tetra substituted at the 3 and 3' positions of the indole
rings. Without limitations as to theory these substitutions prevent the dye from being
an intercalating dye. The identity of the substituents as these positions are not
critical. The SSOMV can in addition have a 3' blocking substituent. Again the chemistry
of the 3' blocking substituent is not critical.
Heterologous Expression
[0066] In certain
examples, heterologous expression is used to express foreign genes or extra copies of endogenous
genes in yeast (for example,
Yarrowia lipolytica)
. Heterologous expression in yeast can be performed using methods well known in the
art. Expression of foreign genes or extra copies of endogenous genes in yeast using
heterologous expression may involve use of a vector that includes (a) promoter sequences
for transcriptional initiation, (b) terminator sequences for termination of transcription,
and (c) a selectable marker. Heterologous expression and expression vectors may be
as described, for example, in
Madzak, C., Gaillardin, C., and Beckerich, J-M., 2004 Heterologous Protein Expression
and Secretion in the Non-Conventional Yeast Yarrowia lipolytica: a review, Journal
of Biotechnology 109:63-81. In certain
examples of the compositions and methods disclosed herein, the vector is pYLEX1 (Yeastern). A non-limiting list of selectable marker
genes that may be used includes
ura3, lys5, trp1, leu2, ade1, E.coli hph encoding hygromycin resistance, and
SUC2 from
Saccharomyces cerevisiae. A non-limiting list of promoters that may be used includes pLEU2, pXPR2, pPOX2, pPOT1,pICL1,
pG3P, pMTP, pTEF, and pRPS7. In certain
examples, the promoter is the hp4d promoter, which is a strong, constitutive hybrid promoter
(
U.S. Patent 6,083,717 issued Jul.4,2000). A non-limiting list of terminator sequences that may be used includes
XPR2t, LIP2t, and
PHO5t.
[0067] In certain
examples, one or more of
Yarrowia lipolytica LYS1 (
Genolevures YALI0B15444g),
TRP1 (
Genolevures YALI0B07667g), and
ADE1 (
Genolevures YALI0E33033g) genes are used as selectable markers. In certain
examples, one or more of
Yarrowia lipolytica URA3 (GenBank: U40564.1) or LEU2 (Genoluveres YALI0C00407) genes arc used as selectable
markers.
[0068] In certain
examples, an integrative expression vector includes one or more promoters and/or terminator
sequences selected from the group consisting of
Yarrowia lipolytica glycolytic pathway genes, alkane or glycerol utilization genes,
XPR2, ACC1, HMG1, ERG1, and
ERG9.
[0069] In certain
examples of one or both subunits of
Yarrowia lipolytica ATP citrate lyase (Genoleveres YALI0D24431g and YALI0E34793g) in
Yarrowia lipolytica are overexpressed.
Modified enzymes
[0070] A modified or mutated enzyme of the present disclosure can be modified or mutated
by base pair changes, insertions, substitutions, and the like.
[0071] The genes encoding enzymes involved in the fatty acid biosynthesis pathway and isoprenoid
biosynthesis pathway arc the preferred targets for mutation. In some examples, the
target gene encodes an acyl CoA carboxylase. In other
examples, the target gene encodes an HMG-CoA reductase. In other
examples, the target gene encodes a squalene epoxidase. In other
examples, the target gene encodes a squalene synthase. In certair
examples, the target gene encodes ATP citrate lyase. Mutations can be designed that reduce
or eliminate the activity of an enzyme, enhance the activity of an enzyme, or that
alter the activity of the enzyme (e.g., change the substrate selectivity).
[0072] In wild-type oleaginous yeast, acetyl-CoA is extensively channeled into fatty acid
biosynthesis via acetyl-CoA carboxylase (ACCase). Thus in order to increase the amount
of acetyl-CoA available for squalene synthesis, it is desirable to reduce the enzymatic
expression or specific activity of ACCase. An exemplary gene sequence for ACCase is
the ACC1 gene in
Saccharomyces cerevisiae as shown in accession number Z71631. Accordingly in certain
examples, reduced intracellular activities of ACCase, the enzyme at the branch point between
mevalonate biosynthesis and triglyceride biosynthesis will decrease the amount of
acetyl-CoA partitioned for oil synthesis, thereby increasing its availability to the
isoprene pathway.
[0073] HMG-CoA reductase activity is the rate-limiting enzyme for isoprene biosynthesis.
Exemplary gene sequences for HMG-CoA reductase include the HMG1 and HMG1 genes in
Saccharomyces cerevisiae as shown in accession numbers NC_001145 and NC_001144, respectfully. Accordingly,
in certain
examples, HMG-CoA reductase activity will be increased by modifying the HMGR gene to increase
transcription, stabilize the resultant protein, and/or reduce product feedback inhibition.
[0074] Decreasing ACCase activity and/or increasing HMG-CoA reductase activity in a yeast
can create a core isoprenoid production organism capable of producing a number of
related isoprenoid products by the manipulation of subsequent enzymes in the pathway.
[0075] Squalene epoxidase catalyzes the first committed step of sterol biosynthesis. An
exemplary gene sequence for Squalene epoxidase is the ERG 1 gene in
Saccharomyces cerevisiae as shown in accession number NC_001139. Accordingly, in certain
examples, squalene epoxidase activity, sensitivity to inhibitors and/or expression will be
attenuated in a yeast, for example by catalytically important residues in the enzyme's
amino acid sequence.
[0076] Squalene synthase catalyzes the synthesis of squalene by condensing two c15 isoprene
precursors (farnesyl diphosphate (FPP)). An exemplary gene sequence for squalene synthase
is the ERG9 gene in
Saccharomyces cerevisiae as shown in accession number NC_001140. Accordingly, in certain examples, squalene
synthase activity and/or expression will be increased in a yeast.
[0077] ATP citrate lyase (E.C. 4.1.3.8) catalytically cleaves citrate to produce acetyl
CoA and oxaloacetate. Acetyl CoA can be used by ACCase for fatty acid biosynthesis
or by acetyl CoA acetyl transferase for the production of isoprenes and derivatives
such as squalene.
[0078] Mevalonate kinase is the first enzyme after HMG-CoA Reductase in the mevalonate pathway,
and catalyzes the conversion of Mevalonate to Mevalonate-5-phosphate. Accordingly,
in certain
examples, mevalonate kinase activity and/or expression levels will be increased in yeast, for
example, by changing catalytically important residues in the enzyme's amino acid sequence
or increasing its gene dosage or transcript levels.
[0079] Glycerol kinase catalyzes the transfer of a phosphate from ATP to glycerol to form
glycerol phosphate. Accordingly, in certain examples, glycerol kinase activity and/or
expression levels will be increased in yeast, for example, by changing catalytically
important residues in the enzyme's amino acid sequence or increasing its gene dosage
or transcript levels.
[0080] The result of the metabolic changes in certain
examples will be to channel carbon from acetyl-CoA to squalene, and attenuate major competitive
pathways for this carbon stream, resulting in a significant increase of squalene produced.
Delivery of gene repair oligonucleobases into yeast cells
[0081] Any commonly known method can be used in the methods
disclosed herein to transform a yeast cell with a gene repair oligonucleobase. Exemplary methods include
the use of electroporation, LiOAc, biolistics, spheroplasting, and/or
Agrobacterium (see, for example,
McClelland, C.M., Chang, Y.C., and Kwon-Chung, K.J. (2005) Fungal Genetics and Biology
42:904-913).
[0082] In certain examples, a gene repair oligonucleobase is introduced into a yeast cell
by electroporation. In some
examples, a gene repair oligonucleobase is introduced into a yeast cell that has been chemically
treated with PEG (3350 or 4000 mw) and/or Lithium Acetate by electroporation. In certain
examples, a gene repair oligonucleobase is introduced into a yeast cell using PEG (3350 or
4000 mw) and/or Lithium Acetate.
[0083] Specific conditions for using microcarriers in the methods
disclosed herein are described in International Publication
WO 99/07865,
US09/129,298. For example, ice cold microcarriers (60 mg/mL), mixed duplex oligonucleotide (60
mg/mL), 2.5 M CaCl
2 and 0.1 M spermidine are added in that order; the mixture gently agitated, e.g.,
by vortexing, for 10 minutes and let stand at room temperature for 10 minutes, whereupon
the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended
in 100% ethanol. Exemplary concentrations of the components in the adhering solution
include 8-10 µg/µL microcarriers, 14-17 µg/µL mixed duplex oligonucleotide, 1.1-1.4
M CaCl
2 and 18-22 mM spermidine. In one example, the component concentrations are 8 µg/µL
microcarriers, 16.5 µg/µL mixed duplex oligonucleotide, 1.3 M CaCl
2 and 21 mM spermidine.
Selection of yeast having the desired modified enzyme
[0085] Yeast expressing the modified enzyme can be identified through any of a number of
means. In one method, a co-conversion strategy using gene repair oligonucleobases
(GRONs) to target both a selectable conversion (i.e., a marker) and a non-selectable
conversion (e.g., a target gene of interest) in the same experiment. In this way,
the cells to which GRONs were not delivered or were unable to transmit the conversions
specified by the GRON would be eliminated. Since delivery of GRONs targeting unrelated
genes is not expected to be selective, at some frequency, a colony with a successfully
selected conversion would also be expected to have a conversion in one of the other
targeted genes. Conversion events would be resolved by single nucleotide polymorphism
(SNP) analysis.
[0086] Thus, genomic DNA is extracted from yeast and screening of the individual DNA samples
using a SNP detection technology, e.g., allele-specific Polymerase Chain Reaction
(ASPCR), for each target. To independently confirm the sequence change in positive
yeast, the appropriate region of the target gene may be PCR amplified and the resulting
amplicon either sequenced directly or cloned and multiple inserts sequenced.
[0087] Alternatively, the incorporation of the mutation into the gene of interest can be
identified by any of a number of molecular biology techniques designed to detect single
nucleotide mutations in extracted nucleic acid (e.g., amplification methods such as
PCR and single nucleotide primer extension analysis). Larger mutations can be detected
by amplification and sequencing of the region of the target gene to be mutated.
[0088] Alternatively, yeast or yeast cells containing the modified enzyme can be identified
by, for example, analysis of the composition of isoprenoids produced by the yeast.
Thus, the yeast can be grown and oils extracted and analyzed using methods known in
the art (e.g., gas chromatography or HPLC).
EXAMPLES
Example 1. Cryptococcus curvatus and Rhodotorula glutinis transformation systems, (not according to the present invention)
[0089] To create a
Cryptococcus curvatus (ATCC strain 20508) and
Rhodotorula glutinis (ATCC strains 10788 and 204091) transformation system, a KANMX expression cassette
(promoter-gene-terminator) which confers kanamycin resistance to
S. cerevisiae is used as a selectable marker to convert the strains from kanamycin sensitivity
to resistance (See
e.g., Baudin, A., et al. (1993) Nucleic Acids Research (21) 3329-3330). The strains are transformed with the expression cassette alone, as well as KANMX
ligated to restriction fragments of a plasmid reported in
R. glutinus (See
e.g Oloke, J.K., and Glick, B.R. (2006) African Journal of Biotechnology 5(4):327-332) containing DNA origins of replication. DNA is introduced into
C.
curvatus and
R. glutinis by electroporation, LiOAc, biolistics, spheroplasting, and/or
Agrobacterium (
McClelland, C.M., Chang, Y.C., and Kwon-Chung, K.J. (2005) Fungal Genetics and Biology
42:904-913).
Example 2. Selectable Markers. (not according to the present invention)
[0090] To generate uracil auxotrophic mutants in
Cryptococcus curvatus and
Rhodotorula glutinis, cells were grown in minimal media containing anti-metabolite 5-fluoroorotic acid
to select for resistant mutants with lesions in the ura3 or ura5 genes. 33 stable
5-FOA
R colonies of
Cryptococcus curvatus and 20 stable 5-FOA
R colonies of
Rhodotorula glutinis were banked. Wild type URA3 genes from both
Cryptococcus curvatus and
Rhodotorula glutinis are cloned and the mutant ura3 genes in the 5-FOA
R isolates are sequenced.
[0091] Other auxotrophic markers are cloned by functional complementation in
Saccharomyces cerevisiae (See
Ho, Y.R., and Chang, M.C. (1988) Chinese Journal of Microbiology and Immunology 21(1):1-8). Genomic and/or cDNA libraries are constructed from
Cryptococcus curvatus and
Rhodotorula glutinis for ligation into a uracil-selectable
Saccharomyces expression vector for transformation into strain YPH500 (
MATα
ura3-52 lys2-801 ade2-101 trp1-Δ
63 his3-Δ
200 leu2-Δ
1) to select for lysine, adenine, tryptophan, histidine, and leucine prototrophs. From
these prototrophs, the corresponding genes for LYS2, ADE2, TRP1, HIS3, and LEU2 are
sequenced from the genomic or cDNA insert.
Example 2. Gene Manipulation in Yeast using RTDS technology. (not according to the present invention)
[0092] The alleles of the leu2, lys5 and ura3 genes from
Yarrowia lipolytica strain ATCC 90811 (leu2-35 lys5-12 ura3-18 XPR2B) were cloned by PCR and their sequences
compared to the wild type alleles to identify differences.
[0093] For ura3, differences were found at positions 1365 (A→G mutation, resulting in a
silent change of AAA→AAG coding for lysine), 1503 (AAGAA extra sequences in ATCC 90811
which results in a frame change, but which comes back in frame at 1511 resulting in
7 additional amino acids, after which the sequence continues as the YL URA3 in GenBank),
1511 (extra T in ATCC 90811), and 1978 (C→T mutation, leading to a stop mutation truncating
the protein 24 amino acids short of the carboxy terminus). A GRON oligonucleotide
was designed to restore prototrophy by converting STOP(TGA)→R (CGA) to yield 264R
based on YlUra3 - YLU40564 amino acid numbering. The GRONs used are YlUra31264/C/40/5'Cy3/3'idC,
which has the sequence VCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGH, and YlUra31264/NC/40/5'Cy3/3'idC,
which has the sequence VCCTCCTCAATAGGATCTCGGTTCTGGCCGTACAGACCTCGH, where V=CY3; H=3'DMT
dC CPG. 10, 30, and 50 µg of cach of the GRONs were transformed into
Yarrowia lipolytica strain ATCC 90811 using a Lithium acetate-based method, and plated onto ura- 2% glucose.
A total of 82 ura+ colonies were obtained with the GRON designed using the coding
strand and 6 colonies with the GRON designed using the non-coding strand, demonstrating
the strand bias common in transforming with gap-repair oligonucleotides. Sequencing
of 18 of the coding-strand transformants demonstrated the intended change in 17 of
the clones.
[0094] For LEU2 differences were found at positions 1710 (extra C absent leading to a frame
shift and premature protein termination); 1896 (extra T); 2036 (T→A mutation, located
after the stop codon); 2177 (extra T in missing, located after stop codon).
[0095] For LEU2 differences were found at positions 1092 (G→A TCG→TCA, a conservative substitution
(Serine)); 1278 (G→A CAG→CAA, a conservative substitution (Glutamine)); 1279 (G→A
GGT→ATT, changing V→I).
[0096] Accordingly, the mutations can be used for various purposes, for example to convert
prototrophic yeast to become auxotrophic and vice versa.
[0097] A similar strategy for demonstrating the effectiveness of RTDS technology in
Cryptococcus curvatus and
Rhodotorula glutinis is performed as described for
Yarrowia lipolytica in which ura3 mutations are corrected to restore prototrophy.
[0098] In certain
examples, the effectiveness of RTDS in
Y.
lipolytica may be demonstrated by integrating a mutated version of the
E. coli hygromycin gene into its genome. This version of the gene, which harbors a point
mutation at G34T, encodes an E12STOP change such that the natural hygromycin sensitivity
of
Y. lipolytica is not affected. Transformation with a GRON correcting this mutation will confer
resistance of the
Y.
lipolytica strain, for example, up to 1000 ug/ml of hygromycin. Double mutations in the hygromycin
resistance (HGH) gene are also constructed, comprising of G34T A37T (E12ASTOP K13STOP)
which may be corrected by a single GRON, and G34T T149G (E12STOP Y46 STOP) which may
be corrected by 2 GRONS.
[0099] To testing GRON activity in
Yarrowia, the natural sensitivity of wild-type
Yarrowia lipolytica to the aminoglycoside antibiotic hygromycin B was used. Hygromycin B (hmB) is an
aminocyclitol antibiotic produced by
Streptomyces hygroscopicus which inhibits protein synthesis in both procaryotes and eucaryotes by interfering
with ribosomal translocation and with aminoacyltRNA recognition. Resistance can be
conferred by introduction of the
hph gene (also known as
aph(4)) from
E. coli (GENBANK V01499) which encodes an aminocyclitol phoshotransferase that inactivates
hygromycin B by covalent addition of a phosphate group to the 4-position of the cyclitol
ring.
Yarrowia lipolytica strain Polg (
Mat a ura3-302::URA3 leu2-270 xpr2-322 axp-2 from Yeastern) was transformed with the
E. coli hph gene containing either a single (E12stop from G34T) or double mutation E12stopK13stop
(G34T.A37T) mutations cloned into vector pyLEX1-2u-ura3-13, putting the gene under
control of the hpd4 promoter and XPR2 terminator. The linearized vector was integrated
into the genome upon selection for restoration of prototrophy conferred by the LEU2
marker. The resultant strains harbor disabled versions of the hygromycin phosphotransferase
gene (hence hygromycin sensitive), and were converted with the following GRONs restoring
either G34T or G34T.A37T to wild type (hygromycin resistant).
GRONs for restoring E12stop to wild type (T34G)
HPH2/C/42/5'Cy3/3 'idC
5'Cy3-GAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAG-3'idC
HPH2/NC/42/5'Cy3/3'idC
5'Cy3-HCTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAGTTC-3'idC
GRONs for restoring E12stopK13stop to wild type (T34G T37A)
HPH3/C/43/5'Cy3/3'idC
5'Cy3-CTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCG-3'idC
HPH3/NC/43/5'Cy3/3'idC
5'Cy3-CGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAG-3'idC
[0100] 30µg of the indicated GRON was used to convert the single- or double- hph mutant
strain in replicate (x6), pooled, and an aliquot plated onto YEPD plates containing
100-1000 µg/ml hygromycin to optimize the signal-to-noise ratio. With both strains,
significant numbers of putativcly converted colonies were obtained at any given hygromycin
concentration above the 'No DNA' control, with a strong bias toward the non-coding
GRON strand in both cases. Taken together, these results suggest GRON conversion of
the hygromycin phosphotransferase gene target in
Yarrowia lipolytica, and further that conversion of two mutations (T34G T37A) can be accomplished using
a single GRON. DNA sequencing is performed to confirm restoration of the wild-type
genotype.
Strain |
DNA |
Colonies on 100 µg/ml Hyeromycin |
Colonies on 200 µg/ml Hygromycin |
Colonies on 400 µg/ml Hygromycin |
Colonies on 600 µg/ml Hygromycin |
Colonies on 800 µg/ml Hygromycin |
Colonies on 1000 µg/ml |
E12stop |
No DNA |
0 |
0 |
1 |
1 |
0 |
0 |
E12stop |
30µg coding strand |
0 |
1 |
6 |
1 |
1 |
0 |
E12stop |
30µg non-coding strand |
20 |
21 |
21 |
12 |
10 |
8 |
E12stopK13stop |
No DNA |
0 |
0 |
4 |
1 |
0 |
0 |
E12stopK13stop |
30µg coding strand |
2 |
2 |
1 |
2 |
0 |
2 |
E12stopK13stop |
30ug non-coding strand |
5 |
3 |
5 |
7 |
8 |
8 |
Example 3. Cloning of target genes. (not according to the present invention)
[0101] The sequences for ACCase, HMGR, squalene synthase and squalene epoxidase, available
in the NCBI database from
Saccharomyces and other yeasts, are used as a source of PCR primers and the corresponding genes
are cloned from
Cryptococcus curvatus and
Rhodotorula glutinis along with their corresponding regulatory regions (promoters, terminators). To identify
'up' and 'down' promoter mutations that increase or decrease transcription, respectively,
the promoters for these four genes are cloned with a relatively error-prone DNA polymerase
to generate point mutations in the promoters, and these fragments are cloned into
plasmids fused with Green Fluorescent Protein (GFP) or beta-galactosidase reporter
genes for testing
in vitro in
S. cerevisiae or
E. coli. Promoter "up" mutations are reintroduced into the HMGR and squalene synthase genomic
sequences by RTDS, while "down" promoter mutations are being made in the genomic ACCase
and squalene epoxidase sequences. The promoters from essential genes (e.g. GAPDH,
actin) in
R. glutinis and
C.
curvatus are cloned for use in heterologous gene expression. Primers for PCR cloning are designed
from homology to these genes in
S.
cerevisiae.
Example 4. Manipulation of target genes for increased squalene production.
[0102] ACCase. The number of copies of the ACCase gene is determined in
R. glutinis and
C.
curvatus and other yeasts. RTDS is utilized to reduce ACCase expression by introducing stop
codons immediately after the translational start site in any extra copies.
[0103] Squalene Epoxidase. Similarly, an increase in squalene accumulation in
S.
cerevisiae has been achieved by disruption of one copy of the squalene epoxidase in the diploid.
Kamimura, N., Hidaka, M., Masaki, H., and Uozumi, T. (1994) Appl. Microb. Biotech.
42: 353-357. The number of copies of squalene epoxidase in
R. glutinis and
C.
curvatus and other yeasts is determined, and RTDS is used to create or insert a stop codon
immediately after the translational start site in extra copies beyond the first one.
[0104] In some
examples, Squalene epoxidase activity is attenuated by addition of terbinafine (an inhibitor
of Squalene epoxidase) to the media. In certain
examples, amino acid changes to the Squalene epoxidase are made to increase the sensitivity
of Squalene epoxidase to terbinafine (for example amino acid changes homologous to
G30S, L37P, and R269G mapped on
Saccharomyces ERG1). In some
examples, the amino acid changes are made by gene synthesis and replacement of the wild-type
gene with the mutant version by homologous recombination. In other
examples, the changes are introduced into the wild-type gene by RTDS.
[0105] HMGR. Both
Saccharomyces cerevisiae and mammalian HMGR enzymes contain amino acid sequences in their linker regions which
are present in many short-lived proteins that are subject to rapid intracellular turnover
in eukaryotes (see
Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234: 364-368; and
Chun, K.T., and Simoni, R.D. (1991) J. Biol. Chem. 267(6): 4236-4246). Similar sequences, if present, are identified in the HMGR genes in
Y.
lipolytica, R. glutinis and/or
C.
curvatus, and eliminated using RTDS to reduce HMGR protein turnover. Such similar sequences
have been found in the
S.
cerevisiae squalene synthase gene, and it is also determined if such sequences are present in
the squalene synthase genes in
Y. lipolytica, R. glutinis and/or
C.
curvatus. The sequences, if present in
Y.
lipolytica, R. glutinis and/or
C.
curvatus squalene synthase, are also eliminated using RTDS to reduce protein turnover.
[0106] HMGR in
S.
cerevisiae comprises two highly conserved domains, of which the N-terminal 552 amino acids are
responsible for membrane association. Overexpression of the truncated HMG1 protein
containing only the C-terminal catalytic portion led a 40-fold increase of HMG-CoA
activity in
S.
cerevisiae with an increased accumulation of squalene to 5.5% of dry matter (
Polakowski, T., Stahl, U., and Lang, C. (1998) Appl. Microbiol. Biotech. 49:66-71). It is determined if
Y.
lipolytica, R. glutinis and
C.
curvatus HMGR proteins have a similar structure, and, if so, fragments having only the soluble
catalytic domain may be expressed.
[0107] The protein structure and DNA sequence of HMGR is highly conserved between eukaryotes
from fungi to mammals, with a membrane-associated N-terminal domain and catalytic
C-terminal domain. The boundary between the two domains can be mapped to a region
of amino acids 500-600 in the
Yarrowia lipolytica HMG1 gene (Genelouvres
Yarrowia lipolytica YALI0E04807g) where the hydrophobicity plot transitions from hydrophobic to hydrophilic.
Resides 548 and 544 are chosen from evaluation of the hydrophobicity plot of
Yarrowia lipolytica HMG1, and its homology to the N-termini of the truncated
Saccharomyces cerevisiae (
Donald, K.A.G., et al, 1997. Appl. Environ. Micro. 63(9): 3341-3344) and
Candida utilis (
Shimada, H. et al, 1998. Appl. Environ. Micro. 64(7):2676-2680) proteins. Accordingly, in one example, amino acids 548-1000 of the C-terminal domain
of
Yarrowia lipolytica HMG1 I is expressed; in a second example amino acids 544-1000 of the C-terminal domain of
Yarrowia lipolytica HVG1 I is expressed. In related examples, amino acids 543-1000 of the C-terminal domain
of
Yarrowia lipolytica HMG1 I is expressed; or amino acids 545-1000 of the C-terminal domains of
Yarrowia lipolytica HMG1 I is expressed; or amino acids 546-1000 of the C-terminal domains of
Yarrowia lipolytica HMG1 I is expressed; or amino acids 547-1000 of the C-terminal domains of
Yarrowia lipolytica HMG1 I is expressed; or amino acids 549-1000 of the C-terminal domains of
Yarrowia lipolytica HMG1 I is expressed.
[0108] Expression of the 457 amino-acid C-terminal catalytic domain of HMGR (residues 543-1000)
in
Y.
lipolytica strain Polg yielded 2% squalene/total lipid compared to 0% in the control strain
containing the vector alone in experiments using shakeflasks. The process is repeated
and expanded using fermenters.
[0109] In Syrian hamsters, activity of the HMGR catalytic domain is down-modulated by phosphorylation
by an AMP-dependent kinase (
Omkumar, R.V., Darnay, B.G., and Rodwell, V.W. (1994) J. Biol. Chem. 269:6810-6814), and a similar mode of regulation has been described in
S.
cerevisiae. It is determined if the HMGR proteins in
R. glutinis, C. curvatus and other yeasts arc similarly regulated, and if so, RTDS is employed to eliminate
the phosphorylation site.
[0110] Squalene synthase. Squalene synthase in mammalian systems is coordinately regulated on the transcriptional
level along with HMG-CoA synthase and farnesyl diphosphate synthase by SREBPs (sterol
regulatory element binding proteins) (
Szkopinsda, A., Swiezewska, E., and Karst, F (2000) Biochem. Biophys. Res. Comm. 267:473-477). SREBPs exist in three forms, of which one binds the squalene synthase promoter.
It is determined if such transcription factors and/or binding sites are present on
the squalene synthase promoter in
R. glutinis, C.curvatus and other yeasts, and, if present, RTDS is used to make changes to such transcription
factors and/or binding sites that enhance transcription of squalene synthase.
[0111] Overexpression of the
Y.
lipolytica Squalene Synthase in
Y.
lipolytica strain Polg yielded 2% squalene/total lipid compared to 0% in the control strain
containing the vector alone using shakeflasks. The process is repeated and expanded
using fermenters.
Example 5. Growth Conditions for Cryptococcus curvatus. (not according to the present invention)
[0112] Cryptococcus curvatus growth was evaluated to determine the best carbon sources to maximize its cell mass
in culture. In a Yeast Extract-based rich media (10 g/L yeast extract, 20 g/L peptone),
C. curvatus grew well in 2-20% w/v glucose, achieving a maximal level of 55 g/L cell dry weight
(CDW) at 16% w/v glucose and above after 4 days. Similarly,
C. curvatus grew in the same media with 3-12% w/v glycerol, achieving a CDW of 40 g/L in 12%
w/v glycerol after 5 days.
C. curvatus was also grown in Biodiesel glycerol (Imperial Western Products, Coachella, CA) up
to 3.5% w/v, resulting in 23 g/L CDW.
Example 6. Environmental manipulation of target genes for increased squalene production.
[0113] Environmental manipulations are tested to increase the net yield of squalene. These
include (a) inhibiting ACCase expression and/or activity with oleic acid, olive or
other vegetable oil(s), inositol, choline, soraphen, fluazifop, and clethodim or other
ACCase inhibiting herbicides, (b) inhibiting squalene epoxidase expression and/or
activity with terbinafine, tolnaftate, and ergosterol or other squalene epoxidase
inhibiting fungicides, (c) manipulating the C/N ratio in glycerol-based media (in
the starting media or by add-ins), (d) varying the nitrogen source in the media (organic
vs. inorganic vs. simple/complex), (e) varying carbon addition regimes (e.g. batch
vs. feeding), (f) examining the effect of depleting nutrients other than carbon source,
(g) varying the carbon source to include mixtures of sugars, sugar alcohols, alcohols,
polyalcohols, and organic acids, (h) selecting for growth on HMGR-inhibitory compounds
such as lovastatin or other statin-type inhibitors, and (i) selecting for high oil
production in culture using lipophillic dyes or stains and/or by analyzing for extractable
lipids using, for example, gravimetric or gas chromatographic methods.
[0114] For example,
Yarrowia lipolytica ATCC 90904 was cultivated in high Carbon/Nitrogen ratio media (C/N = 420,
Li, Y-H., Liu, B., Zhao, Z-B., and Bai, F-W. 2006 "Optimized Culture Medium and Fermentation
Conditions for Lipid Production by Rhodosporidium toruloides" Chinese Journal of Biotechnology
22(4): 650-656) (hereinafter "CYM001 Media") supplemented with 0 to 50 µg/ml terbinafine at 30'C,
300 rpm for 120 h. Concentrations of 12.5µg/ml or higher of terbinafine resulted in
up to 18.5% of total lipid as squalene.
[0115] Various
Yarrowia lipolytica strains are used for lipid and squalene production including ATCC 20688, ATCC 90811,
ATCC 90904, ATCC 90812, ATCC MYA-2613, and Yeastern polg. For example,
Yarrowia lipolytica strain
polg (Yeastern) was cultivated in high Carbon/Nitrogen ratio media (C/N = 420,
Li, Y-H., Liu, B., Zhao, Z-B., and Bai, F-W. 2006 "Optimized Culture Medium and Fermentation
Conditions for Lipid Production by Rhodosporidium toruloides" Chinese Journal of Biotechnology
22(4): 650-656) (hereinafter "CYM001 Media") supplemented with 0 to 50 µg/ml terbinafine at 30'C,
300 rpm for 120 h. Concentrations of 12.5µg/ml or higher of terbinafine resulted in
up to 38 % of total lipid as squalene and Values of total lipid/Cell Dry weight of
up to 51% were achieved.
[0116] In another example,
Yarrowia lipolytica ATCC 90904 was cultivated in CYM001 media supplemented with 0 to 50 µg/ml Oleic acid
at 30'C, 300 rpm for 120 h. Supplementation with 10µl/ml Oleic acid was found to improve
lipid accumulation 10-fold in lipid/CDW (cell dry weight) over no supplementation.
[0117] In a further example,
Yarrowia lipolytica ATCC 90904 was cultivated in CYM001 media supplemented with 0 to 200 µM clethodim
at 30'C, 300 rpm for 120 h. Supplementation of 200 µM clethodim resulted in a 60-fold
increase in the yield (mg) of squalene per 60-ml flask.
[0118] Increased oxygen has been shown to cause the differential regulation of HMG1 and
HMG2 in
S.
cerevisiae, resulting in rapid degradation of HMG2 and increased expression of HMG1 under aerobic
conditions (
Casey, W.M., Keesler, G.A., Parks, L.W. (1992) J. Bact. 174:7283-7288). It is determined if the number of HMGR genes in our oleaginous yeasts is affected
by oxygen and, if so, their expression and activity is manipulated in the fermenter
by altering oxygen levels.
[0119] Starting with "CYM001 Media" (
Li, Y-H., Liu, B., Zhao, Z-B., and Bai, F-W. (2006) Chinese Journal of Biotechnology
22(4):650-656), various components and concentrations of components are changed (including the
addition of new components) to improve cell growth, percent total lipid contcnt/unit
mass of cells, and percent squalene/total lipid. Media components that are evaluated
include: carbon sources: glycerol, glucose, nitrogen sources: ammonium compounds,
nitrates, amino acids, mineral salts: potassium, magnesium, sodium, iron, manganese,
zinc, calcium, copper, yeast extract, lipid precursors and lipid synthesis affectors:
terbinafine, clethodim, oleic acid, palmitoleic acid, linoleic acid, linolenic acid
and antifoaming agents. Other factors that are evaluated include: percent inoculum,
elapsed fermentation time, temperature, pH, back pressure, dissolved oxygen (DO),
feed composition, feed strategy and agitation strategy.
Example 7. Strain Selection. (not according to the present invention)
[0120] Traditional strain selection methods are used in oleaginous yeasts to increase their
net squalene productivity. Strains mutagenized by UV, nitrosoguanidine, or ethane
methyl sulfonate are screened and/or selected for increased squalene accumulation.
Strains are also subjected to iterative selection pressure, such as repeated passage
on YEP (15 g/L yeast extract, 5 g/L peptone) media containing 3% glycerol or media
containing lovastatin and other known HMGR inhibitors. Strains are also subjected
to repeated passage on CYM001 Media containing varying amounts of glycerol and/or
glucose or media containing lovastatin and/or other known HMGR inhibitors, and/or
squalene synthase inhibitors to obtain spontaneous mutants with increased HMGR and/or
squalene synthase activity. Such mutations may be in HMGR, squalene synthase, or other
genes ("secondary site mutations").
[0121] Unless otherwise defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which this
invention belongs.
[0122] The inventions illustratively described herein may suitably be practiced in the absence
of any element or elements, limitation or limitations, not specifically disclosed
herein. Thus, for example, the terms "comprising," "including," "containing," etc.
shall be read expansively and without limitation. Additionally, the terms and expressions
employed herein have been used as terms of description and not of limitation, and
there is no intention in the use of such terms and expressions of excluding any equivalents
of the features shown and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the invention claimed.
[0123] Thus, it should be understood that although the invention has been specifically disclosed
by preferred embodiments and optional features, modification, improvement and variation
of the inventions embodied therein herein disclosed may be resorted to by those skilled
in the art, and that such modifications, improvements and variations are considered
to be within the scope of the appended claims. The materials, methods, and examples
provided here are representative of preferred embodiments, are exemplary, and are
not intended as limitations on the scope of the invention.
SEQUENCE LISTING
[0124]
<110> CIBUS OILS, LLC
<120> METHODS AND COMPOSITIONS FOR PRODUCING SQUALENE USING YEAST
<130> 095142-0550
<140> PCT/US2010/057668
<141> 2010-11-22
<150> 61/263,775
<151> 2009-11-23
<160> 6
<170> PatentIn version 3.5
<210> 1
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